Inactivation of cytochrome P4502B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables

A series of arylalkyl isothiocyanates were evaluated for their ability to i nactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl is othiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P45 0) enzymes has been implicated in their chemopreventative abilities. The na turally occurring isothiocyanates BITC and PEITC inactivated P450 2BI in a time-and concentration-dependent manner, whereas the synthetic isothiocyana tes phenylpropyl and phenylhexyl isothiocyanate did not result in inactivat ion, but were potent competitive inhibitors of P450 2BI activity. The kinet ics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4- (trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivat ed in a mechanism-based manner. The loss of O-deethylation activity followe d pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K-I) was 5.8 muM, and the maximal rate constant for inactivation was 0.66 min(-1) at 23 degreesC . BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost wi thin 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption s pectrum of the inactivated protein did not decrease significantly, and subs equent HPLC analysis indicated no apparent modification of the heme. HPLC a nd protein precipitation analyses indicated that the P450 apoprotein was co valently modified by a metabolite of BITC. Determination of the binding sto ichiometry indicated that 0.90 +/- 0.16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactiva tor of P450 2B1 and that inactivation occurs primarily through protein modi fication.